Abstract: Klebsiella pneumoniae is the significant opportunistic pathogens from Enterobacteriaceae family which cause both community (non-hospital) and hospital-acquired infections. Our study focuses on the molecular characterization of the colistin and tigecycline-resistant K. pneumoniae and its virulence from Tamil Nadu. A total of 315 clinical samples were collected from Trichy, Madurai, and Chennai, from which 93 K. pneumoniae were isolated. On subjecting to the antibiotic susceptibility testing, it was found that 83% were MDR. It was studied that 62% were resistant to colistin, and all the isolates were susceptible to tigecycline by Minimum Inhibitory concentration (MIC) broth dilution. Based on the Epsilometer test, the tigecycline heteroresistant (HR) isolates were identified with three subpopulations on the resistant zone. All the K. pneumoniae isolates, including the subpopulation (n=96), were subjected for detecting hypermucoviscous (HMV) phenotype using string test and biofilm production using crystal violet staining revealed that 22/96 K. pneumoniae isolates were HMV and strong (n=25), moderate (n=34), weak (n=20) and no (n=17) BFPs (BFP). The molecular studies revealed that there were no mcr genes, class 1 integron (n=23) with devoid of gene cassette, capsular typing K1 (n=5), K2 (n=7), K5 (n=2), Virulence genes namely rmpA (n=5), aerobactin (n=12), KfuBC (n=12) were identified using multiplex polymerase chain reaction (PCR). Based on the molecular characterization, we observed 88 isolates were classical K. pneumoniae (cKP), and 8 isolates were hypervirulent K. pneumoniae (hvKP). In vivo studies were carried out using Galleria mellonella in order to access pathogenicity of cKP and hvKP. The HMV hvKP strains were highly pathogenic among the hvKP strains, and the non-K1/K2 and K2 strains were pathogenic among the cKP strains. The whole-genome shotgun sequencing for the tigecycline heteroresistant K. pneumoniae and the subpopulation that maintained stability was performed using oxford Nanopore MinIon reveals that they are extensive drug-resistant (XDR) by analysing the sequence using Resfinder version 4.0 and are found to be cKP strains using the virulence finder version 2.0. Our data revealed the emergence of CL-R hvKP strains with increased virulence; the tigecycline heteroresistance was identified and characterized at the genomic level for the first time in Tamil Nadu, India.